Project Summary/Abstract It has been challenging to find and validate molecular targets for extending human healthspan because most clinical biomarkers are neither sufficiently mechanistic nor proximal to fundamental mechanisms of aging to serve as indicators. To address the challenge of developing biomarkers of aging, we will primarily focus on epigenetic alterations because it is highly likely that with this work DNA methylation (DNA) will come out as a valid biomarker ready for clinical application. The overarching goals of this proposal are a) to realize the great promise of DNAm based biomarkers of aging (known as epigenetic clocks) for human interventional studies and b) to advance their mechanistic understanding. This proposal builds on our active research program surrounding DNAm based biomarkers of aging and their relationship to markers of cellular senescence. We and others have demonstrated that existing prototypes of DNAm biomarkers are predictive of lifespan and many age-related conditions. We have established that some DNAm biomarkers relate to lifestyle interventions and existing DNAm biomarkers are already being used in human clinical trials of anti-aging interventions. With these proof-of-concept studies completed, we enter the next phase where these biomarkers need to be optimized to track the effectiveness of interventions in human studies. Although it is widely acknowledged that DNAm biomarkers are remarkably robust, they remain sensitive to technical variation. To minimize such spurious variation, we will optimize all components within the workflow, from collection of human samples to the final point of analysis. In our previous planning grant, our network of researchers designed a study for evaluating the utility of DNAm based biomarkers of aging in human longitudinal cohort studies. Building on these plans, we will test whether DNAm biomarkers are indicators of a fundamental aging process underlying healthspan by carrying out human longitudinal cohort studies, genetic studies, and interventional studies. We will relate DNAm biomarkers to markers of cellular senescence and a battery of clinical biomarkers of aging in order to advance mechanistic insights. We will evaluate whether a panel of biomarkers is more predictive of healthspan/multiple health outcomes as opposed to a single marker. We will optimize DNAm biomarkers for use in human ex vivo studies for testing responsiveness to various anti-aging or pro-aging interventions. The resulting optimized system also serves as a potential high-throughput drug screening system, which will have the advantage of being controlled for potential confounding factors and is of human origin. Collectively, these investigations will result in (a) the optimization of the entire workflow process for the application of DNAm biomarkers in human interventional studies and (b) context-of-use statements that fully describe the purpose and use of each biomarker.